Genomic sequences for protein production and delivery

ABSTRACT

An isolated nucleic acid molecule that hybridizes under stringent conditions, or shares at least 80% sequence identity, with a defined genomic region upstream of the coding region of a FSHβ gene, and a DNA construct containing that nucleic acid molecule as a targeting sequence for homologous recombination.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/084,663, filed May 7, 1998, herein incorporatedby reference.

FIELD OF THE INVENTION

[0002] This invention relates to genomic DNA.

BACKGROUND OF THE INVENTION

[0003] Current approaches to treating disease with therapeutic proteinsinclude both administration of proteins produced in vitro and genetherapy. In vitro production of a protein generally involves theintroduction of exogenous DNA coding for the protein of interest intoappropriate host cells in culture. Gene therapy methods, on the otherhand, involve administering to a patient genetically engineered cells,plasmids, or viruses that contain a sequence encoding the therapeuticprotein of interest.

[0004] Certain therapeutic proteins may also be produced by altering theexpression of their endogenous genes in a desired manner with genetargeting techniques. See, e.g., U.S. Pat. Nos. 5,641,670, 5,733,761,and 5,272,071; WO 91/06666; WO 91/06667; and WO 90/11354, all of whichare incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0005] The present invention is based upon the identification andsequencing of genomic DNA 5′ to the coding sequences of the humanfollicle-stimulating hormone β (“FSHβ”) gene. This DNA can be used, forexample, in a DNA construct that alters (e.g., increases) expression ofan endogenous FSHβ gene in a mammalian cell upon integration into thegenome of the cell via homologous recombination. “Endogenous FSHβ gene”refers to a genomic (i.e., chromosomal) copy of a gene that encodesFSHβ. The construct contains a targeting sequence including or derivedfrom the newly disclosed 5′ noncoding sequence, and a transcriptionalregulatory sequence. The transcriptional regulatory sequence preferablydiffers in sequence from the transcriptional regulatory sequence of theendogenous FSHβ gene. The targeting sequence directs the integration ofthe regulatory sequence into a region within or upstream of theFSHβ-coding sequences of the target gene such that the regulatorysequence becomes operatively linked to the endogenous coding sequence.By “operatively linked” is meant that the regulatory sequence can directexpression of the endogenous FSHβ-coding sequence. The construct mayadditionally contain a selectable marker gene to facilitate selection ofcells that have stably integrated the construct, and/or another codingsequence operatively linked to a promoter.

[0006] In one embodiment, the DNA construct contains: (a) a targetingsequence, (b) a regulatory sequence, (c) an exon, and (d) a splice-donorsite. The targeting sequence directs the integration of itself andelements (b)-(d) into a region within or upstream of the FSHβ-codingsequences of the target gene. Once integrated, element (b) can directtranscription of elements (c) and (d) and all downstream codingsequences of the endogenous gene. In the construct, the exon isgenerally 3′ of the regulatory sequence, and the splice-donor site is atthe 3′ end of the exon.

[0007] In another embodiment, the DNA construct comprises: (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of itself andelements (b)-(f) such that elements (b)-(f) are within or upstream ofthe endogenous gene. The regulatory sequence then directs production ofa transcript that includes not only elements (c)-(f), but also theendogenous FSHβ coding sequence. Preferably, the intron and thesplice-acceptor site are situated in the construct downstream from thesplice-donor site.

[0008] The targeting sequence is homologous to a pre-selected targetsite in the genome with which homologous recombination is to occur. Itcontains at least 20 (e.g., at least 30, 50, 100, or 1000) contiguousnucleotides from SEQ ID NO:4, which corresponds to nucleotides −7454 to−1417 of human FSHβ genomic sequence (numbering relative to thetranslation start site), or SEQ ID NO:5, which corresponds tonucleotides −696 to −155 of human FSHβ genomic sequence. By “homologous”is meant that the targeting sequence is identical or sufficientlysimilar to its genomic target site so that the targeting sequence andtarget site can undergo homologous recombination within a human cell. Asmall percentage of basepair mismatches is acceptable, as long ashomologous recombination can occur at a useful frequency. To facilitatehomologous recombination, the targeting sequence is preferably at leastabout 20 (e.g., at least 50, 100, 250, 400, or 1,000) base pairs (“bp”)long. The targeting sequence can also include genomic sequences fromoutside the region covered by SEQ ID NO:4 or 5, so long as it includesat least 20 nucleotides from within one of the two regions. For example,additional targeting sequence could be derived from the sequence lyingbetween SEQ ID NO:4 and the transcription initiation sequence of theFSHβ gene.

[0009] Due to polymorphism that may exist at the FSHβ genetic locus,minor variations in the nucleotide composition of any given genomictarget site may occur in any given mammalian species. Targetingsequences that correspond to such polymorphic variants (particularlyhuman polymorphic variants) of SEQ ID NO:4 or 5 are within the scope ofthis invention.

[0010] Upon homologous recombination, the regulatory sequence of theconstruct is integrated into a pre-selected region upstream of thecoding sequence of a FSHβ gene in a chromosome of a cell. The resultingnew transcription unit containing the construct-derived regulatorysequence alters the expression of the target FSHβ gene. The FSHβ proteinso produced may be identical in sequence to the FSHβ protein encoded bythe unaltered, endogenous gene, or may contain additional, substituted,or fewer amino acid residues as compared to the wild type FSHβ protein,due to changes introduced as a result of homologous recombination.

[0011] Altering gene expression encompasses activating (or causing to beexpressed) a gene which is normally silent (i.e., essentiallyunexpressed) in the cell as obtained, increasing or decreasing theexpression level of a gene, and changing the regulation pattern of agene such that the pattern is different from that in the cell asobtained. “Cell as obtained” refers to the cell prior to homologousrecombination.

[0012] Also within the scope of the invention is a method of using thepresent DNA construct to alter expression of an endogenous FSHβ gene ina mammalian cell. This method includes the steps of (i) introducing theDNA construct into the mammalian cell, (ii) maintaining the cell underconditions that permit homologous recombination to occur between theconstruct and a genomic target site homologous to the targetingsequence, to produce a homologously recombinant cell; and (iii)maintaining the homologously recombinant cell under conditions thatpermit expression of the FSHβ-coding sequence under the control of theconstruct-derived regulatory sequence. At least a part of the genomictarget site is 5′ to the coding sequence of an endogenous FSHβ gene.That is, the genomic target site can contain coding sequence as well as5′ non-coding sequence.

[0013] The invention also features transfected or infected cells inwhich the construct has undergone homologous recombination with genomicDNA upstream of the endogenous ATG initiation codon in one or bothalleles of the endogenous FSHβ gene. Such transfected or infected cells,also called homologously recombinant cells, have an altered FSHβexpression pattern. These cells are particularly useful for in vitroFSHβ production and for delivering FSHβ via gene therapy. Methods ofmaking and using such cells are also embraced by the invention. Thecells can be of vertebrate origin such as mammalian (e.g., human,non-human primate, cow, pig, horse, goat, sheep, cat, dog, rabbit,mouse, guinea pig, hamster, or rat) origin.

[0014] The invention further relates to a method of producing amammalian FSHβ protein in vitro or in vivo by introducing theabove-described construct into the genome of a host cell via homologousrecombination. The homologously recombinant cell is then maintainedunder conditions that allow transcription, translation, and optionally,secretion of the FSHβ protein.

[0015] The invention also features isolated nucleic acids comprising asequence of at least 20 (e.g., at least 30, 50, 100, 200, or 1000)contiguous nucleotides of SEQ ID NO:4, or at least 20 (e.g., at least30, 50, 100, or 200) contiguous nucleotides of SEQ ID NO:5, or of asimilar-sized portion of a sequence identical to SEQ ID NO:4 or 5 exceptfor polymorphic variations or other minor variations (e.g., less than 5%of the sequence) which do not prevent homologous recombination with thetarget sequence.

[0016] In one embodiment, the isolated nucleic acid of the inventionincludes a contiguous 100 bp block of SEQ ID NO:4 or 5. For example, theisolated DNA can contain nucleotides 1 to 100, 101 to 200, 201 to 300,301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900,901 to 1000, 1001 to 1100, 1101 to 1200, 1201 to 1300, 1301 to 1400,1401 to 1500, 1501 to 1600, 1601 to 1700, 1701 to 1800, 1801 to 1900,1901 to 2000, 2001 to 2100, 2101 to 2200, 2201 to 2300, 2301 to 2400,2401 to 2500, 2501 to 2600, 2601 to 2700, 2701 to 2800, 2801 to 2900,2901 to 3000, 3001 to 3100, 3101 to 3200, 3201 to 3300, 3301 to 3400,3401 to 3500, 3501 to 3600, 3601 to 3700, 3701 to 3800 3801 to 3900,3901 to 4000, 4001 to 4100, 4101 to 4200, 4201 to 4300, 4301 to 4400,4401 to 4500, 4501 to 4600, 4601 to 4700, 4701 to 4800, 4801 to 4900,4901 to 5000, 5001 to 5100, 5101 to 5200, 5201 to 5300, 5301 to 5400,5401 to 5500, 5501 to 5200, 5601 to 5700, 5701 to 5800, 5801 to 5900,5901 to 6000, or 5939 to 6038 of SEQ ID NO:4 or its complement.Alternatively, the isolated nucleic acid of the invention can includenucleotides 1 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, or443 to 542 of SEQ ID NO:5. These blocks of SEQ ID NO:4 or 5 or theircomplements are useful as targeting sequences in the constructs of theinvention.

[0017] In the isolated DNA, the contiguous nucleotide sequence is notlinked to a sequence encoding full-length FSHβ, or at least not linkedin the same configuration (i.e., separated by the same sequence) as inany native genome. The term “isolated DNA”, as used herein, thus doesnot denote a chromosome or large piece of genomic DNA (as might beincorporated into a cosmid or yeast artificial chromosome) that includesnot only part or all of SEQ ID NO:4 or 5, but also an intact FSHβ-codingsequence and all of the sequence which lies between the FSHβ codingsequence and the sequence corresponding to SEQ ID NO:4 or 5 as it existsin the genome of a cell. It does include, but is not limited to, a DNA(i) which is incorporated into a plasmid or virus; or (ii) which existsas a separate molecule independent of other sequences, e.g., a fragmentproduced by polymerase chain reaction (“PCR”) or restrictionendonuclease treatment. The isolated DNA preferably does not contain asequence which encodes intact FSHβ precursor (i.e., FSHβ complete withits endogenous secretion signal peptide).

[0018] The invention also includes isolated DNA comprising a strandwhich contains a sequence that is at least 100 (e.g., at least 200, 400,or 1000) nucleotides in length and that hybridizes under either highlystringent or moderately stringent conditions with SEQ ID NO:4 or 5, orthe complement of SEQ ID NO:4 or 5. The sequence is not linked to aFSHβ-coding sequence, or at least not linked in the same configurationas occurs in any native genome. By moderately stringent conditions ismeant hybridization at 50° C. in Church buffer (7% SDS, 0.5% NaHPO₄, 1 MEDTA, 1% bovine serum albumin) and washing at 50° C. in 2X SSC. Highlystringent conditions are defined as hybridization at 42° C. in thepresence of 50% formamide; a first wash at 65° C. with 2X SSC containing1% SDS; followed by a second wash at 65° C. with 0.1X SSC.

[0019] Also embraced by the invention is isolated DNA comprising astrand which contains a sequence that (i) is at least 100 (e.g., atleast 200, 400, or 1000) nucleotides in length and (ii) shares at least80% sequence (e.g., 85%, 90%, 95%, or 98%) identity with a segment ofequal length from SEQ ID NO:4 or 5, or from the complement of SEQ IDNO:4 or 5. The sequence is not linked to a FSHβ-coding sequence, or atleast not linked in the same configuration as occurs in any nativegenome.

[0020] Where a particular polypeptide or nucleic acid molecule is saidto have a specific percent identity or conservation to a referencepolypeptide or nucleic acid molecule, the percent identity orconservation is determined by the algorithm of Myers and Miller, CABIOS(1989), which is embodied in the ALIGN program (version 2.0), or itsequivalent, using a gap length penalty of 12 and a gap penalty of 4where such parameters are required. All other parameters are set totheir default positions. Access to ALIGN is readily available. See,e.g., http://www2.igh.cnrs.fr/bin/align-guess.cgi on the Internet.

[0021] The invention also features a method of delivering FSHβ to ananimal (e.g., a mammal such as a human, non-human primate, cow, pig,horse, goat, sheep, cat, dog, rabbit, mouse, guinea pig, hamster, orrat) by providing a cell whose endogenous FSHβ gene has been activatedas described herein, and implanting the cell in the animal, where thecell secretes FSHβ. Also included in the invention is a method ofproducing FSHβ by providing a cell whose endogenous FSHβ gene has beenactivated as described herein, and culturing the cell in vi tro underconditions which permit the cell to express and secrete FSHβ.

[0022] The isolated DNA of the invention can be used, for example, as asource of an upstream PCR primer for use (when combined with a suitabledownstream primer) in obtaining the regulatory and/or coding regions ofan endogenous FSHβ gene, or as a hybridization probe for indicating thepresence of chromosome 11 in a preparation of human chromosomes. It canalso be used, as described below, in a method for altering theexpression of an endogenous FSHβ gene in a vertebrate cell.

[0023] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Exemplary methods andmaterials are described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

[0024] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a schematic diagram showing the genomic structure of thehuman FSHβ gene.

[0026]FIG. 2 is a schematic diagram showing the genomic region of thehuman FSHβ gene (top) encompassed by the insert (bottom) of plasmidpHFB2. The three bars in the middle represent genomic regions of thegene whose sequences have been published.

[0027]FIG. 3 a representation of a partial sequence (SEQ ID NO:1) of ahuman FSHβ gene, including 7,454 nucleotides of the sequence 5′ to theATG initiation codon. Also shown is a partial polypeptide sequence (SEQID NO:2) encoded by the coding sequence. Published sequences areunderlined. “SD” and “SA” stand for splice-donor site andsplice-acceptor site, respectively. “Mature” denotes the beginning of amature FSHβ protein.

[0028]FIG. 4 is a schematic diagram showing a construct of theinvention. The construct contains a first targeting sequence (1); anamplifiable marker gene (AM); a selectable marker gene (SM); aregulatory sequence; a CAP site; a sequence identical to the first,non-coding exon of a human FSHβ gene; an unpaired splice-donor site(SD); and a second targeting sequence (2). The black boxes representcoding DNA and the stippled boxes represent untranslated sequences.

[0029] FIGS. 5-7 are schematic diagrams illustrating three constructs ofthe invention. The constructs differ in the size of the aldolase 5′ UTSinserted into the plasmid. These constructs include a sequence encodinga glycoprotein α-subunit (i.e, FSHα) linked to a cytomegalovirus (“CMV”)promoter. The abbreviations shown in the figure are: “UTS” foruntranslated sequence; “amp” for ampicillin; “Ori” for replicationorigin,; “SD” for splice-donor site; “HSV TK” for herpes simplex virusthymidine kinase gene; “DHFR” for dihydrofolate reductase; “HBV” forhepatitis B virus; and “hGH” for human growth hormone.

[0030]FIG. 8 is a bar graph of FSH production from HT-1080 cellstransfected with pGA308 (FIG. 5) and selected for growth in the presenceof various concentrations of methotrexate.

[0031]FIG. 9 is a representation of SEQ ID NO:4, a sequence upstream ofa human FSHβ transcription start site.

[0032]FIG. 10 is a representation of SEQ ID NO:5, a sequence upstream ofa human FSHβ transcription start site.

[0033]FIG. 11 is a representation of a first targeting sequence (SEQ IDNO: 6) used in a construct of the invention.

[0034]FIG. 12 is a representation of a second targeting sequence (SEQ IDNO: 5) used in a construct of the invention.

DETAILED DESCRIPTION

[0035] The present invention is based on the discovery of the nucleotidecomposition of sequences upstream to the coding sequence of a human FSHβgene.

[0036] FSH is a gonadotrophin which plays an essential role in themaintenance and development of oocytes and spermatozoa in normalreproductive physiology. FSH possesses two subunits, α and β, the latterbeing responsible for FSH's biological specificity.

[0037] The human FSHβ gene encodes a 129 amino acid precursor proteincontaining a 16 amino acid signal peptide. The gene contains three exonsand two introns, with the first exon being a non-coding exon. Thegenomic map of the human FSHβ gene is shown in FIG. 1. The map isconstructed based on published sequences (HUMFSHBQ1, GenBank accessionnumbers M54912, M38644, M21219, and M18536) that correspond to threeseparate genomic segments (FIG. 1). The first segment is 720 bp long andcontains 530 bp of nontranscribed upstream sequences, exon 1 (63 bp;non-coding), and 127 bp of intron 1. The second segment begins atposition −152 and ends at position +367 (all positions referred toherein are relative to the translational initiation site, unlessspecified otherwise). This segment includes 146 bp of intron 1, exon 2(165 bp), and 208 bp of intron 2. The third segment contains 102 bp ofintron 2 and exon 3, and extends 1,480 bp past the translational stopcodon.

[0038] Specific Sequences 5′ to a FSHβ Coding Sequence and Their Use inAltering Endogenous FSHβ Gene Expression

[0039] To obtain genomic DNA containing sequence upstream to a FSHβgene, a human leukocyte genomic library in lambda EMBL3 (Clontechcatalog # HL1006d) was screened with a 40 bp oligonucleotide probe,BETA2. This probe is derived from 23 bp of exon 1 and 17 bp of intron 1,and has the following sequence:

[0040] 5′ TTGGCATCTACCGTTTTCAAGTGGTGACAGCTACTTTTGA 3′ (SEQ ID NO:3)

[0041] Approximately one million recombinant phage were screened withthe radiolabelled BETA2 probe. One phage plaque, designated clone8-1-1-1, was isolated. The 7.6 kb HindIII-KpnI fragment from phage8-1-1-1 was subcloned into pBluescript II SK+ (Stratagene, La Jolla,Calif.) to produce a plasmid containing about 6.6 kb of upstreamsequences, exon 1, intron 1, exon 2, and 9 bp of intron 2 (FIG. 2). Theplasmid was designated pHFB2.

[0042] The pHFB2 plasmid was sequenced by the Sanger method. Thesequence data sets were aligned to obtain the complete sequence of theentire phage 8-1-1-1 insert. This nucleotide sequence (SEQ ID NO:1) isshown in FIG. 3.

[0043] The insert was shown to encompass a 7,622 bp region of the FSHβgene, starting at position −7,454 (FIG. 3). The sequences encompassingpositions −7,454 to −1,417 (6,038 bp of the upstream sequence; SEQ IDNO:4) and positions −696 to −155 (542 bp of intron 1; SEQ ID NO:5) havenot been reported previously.

[0044] To alter the expression of an endogenous FSHβ gene, the generalapproach shown in FIG. 4 was used. Nucleotides 3860 to 5784 of SEQ IDNO:4 served as the first (5′) targeting sequence, while SEQ ID NO:5served as the second (3′) targeting sequence. DNA fragments containingthese sequences were then subcloned into plasmids to produce targetingconstructs pGA308, pGA301, and pGA307, which are illustrated in FIGS.5-7, respectively. These plasmids each contain about a 3.2 Kb 5′targeting sequence and about a 0.5 Kb 3′ targeting sequence.

[0045] HT-1080 cells were separately transfected with each of theplasmids and placed under G418 selection. After approximately 14 days,G418 resistant colonies in 6-well plates were counted. In addition, theconditioned medium in each well was screened for GA-FSH expression byELISA. Cells exhibiting GA-FSH production were trypsinized and counted.The cells were then diluted and plated in 96-well plates to generateclones. After about two weeks of culture, clonal cell populations werescreened for GA-FSH production by ELISA. Colonies found to produceGA-FSH were expanded in culture and stored or further analyzed. Table 1summarizes the endogenous gene activation frequency and otherobservations from the above cloning procedure.

[0046] The cells transfected with pGA308 were studied in more detail.FIG. 8 indicates the range of FSH production achieved inpGA308-transfected HT1080 cells cultured in media having variousconcentrations of methotrexate. The bar labeled “0.2 (cloned)”represents the FSH production from a cell line cloned by limitingdilution of cells resistant to 0.2 μM methotrexate. The results graphedin FIG. 8 clearly indicate that higher concentrations of methotrexatecan yield cell lines that produce at least 50 μg/10⁶ cells in a day.TABLE 1 Average FSHβ Total No. Total No. Acti- Total No. ProductionPlasmid G418- FSHβ Gene vation Clonal Cell (ng/10⁶ Trans- ResistantActivation Fre- Lines cells in fected Colonies Events quency Isolated 24hours) pGA301 38012 3 1/12671 11 465 pGA307 31068 3 1/10356 20 450pGA308 27474 4 1/6869 16 521

General Methodologies Alteration of Endogenous FSHβ Expression

[0047] Using the above-described FSHβ upstream sequences, one can alterthe expression of an endogenous human FSHβ gene by a method as generallydescribed in U.S. Pat. No. 5,641,670. One strategy is shown in FIG. 4.In this strategy, a targeting construct is designed to include a firsttargeting sequence homologous to a first target site upstream of thegene, an amplifiable marker gene, a selectable marker gene, a regulatoryregion, a CAP site, an exon, an unpaired splice-donor site, and a secondtargeting sequence corresponding to a second target site downstream ofthe first target site, and terminating either within or upstream of theFSHβ-coding sequence. In this strategy, the first and second targetsites are immediately adjacent in the chromosome prior to homologousrecombination, but such configuration is not required (see also below).Homologously recombinant cells will produce an mRNA precursor whichcorresponds to the exogenous exon and splice-donor site, and anysequence between the splice donor site and the transcription terminationsequence of the FSHβ gene, including the FSHβ introns, exons, and 3′untranslated region (FIG. 4). Splicing of this message results in a mRNAin which the exogenous exon is fused to exon 2 of the endogenous FSHβgene. Translation of the mRNA produces a precursor FSHβ.

[0048] Other approaches can also be employed. For example, the firstand/or second target sites can be in the first intron of the FSHβ gene.Alternatively, the DNA construct may be designed to include, from 5′ to3′, a first targeting sequence, an amplifiable marker gene, a selectablemarker gene, a regulatory region, a CAP site, an exon, a splice-donorsite, an intron, a splice-acceptor site, and a second targetingsequence. For this strategy, the 5′ end of the second target site ispreferably less than 40 bp upstream of the normal FSHβ transcriptionalstart site, in order to avoid undesired ATG start codons. A mRNAprecursor produced from the homologously recombined locus will includethe exogenous exon, the exogenous splice-donor site, the exogenousintron, the exogenous splice-acceptor site, and any sequences betweenthe exogenous splice acceptor site and the transcription terminationsite of the endogenous FSHβ gene. Splicing of this transcript willgenerate a mRNA which can be translated to produce a precursor of humanFSHβ, having either the normal FSHβ secretion signal sequence or agenetically engineered secretion signal sequence. The size of theexogenous intron and thus the position of the exogenous regulatoryregion relative to the coding region of the endogenous gene can bevaried to optimize the function of the regulatory region.

[0049] In any activation strategy, the first and second target sitesneed not be immediately adjacent or even be near each other. When theyare not immediately adjacent to each other, a portion of the FSHβ gene'snormal upstream region and/or a portion of the coding region would bedeleted upon homologous recombination.

[0050] If desired, the product of the activated FSHβ gene can beproduced in a cell type that expresses a human glycoprotein α-subunit(FSHα) gene, the product of which forms a heterodimer with the productof the FSHβ gene. This may be a naturally occurring cell strain or cellline. Alternatively, the human glycoprotein α-subunit gene (Genbanksequence HUMGLYCA1) can be co-expressed with the product of the FSHβgene, with such co-expression accomplished by expression of the humanglycoprotein α-subunit gene or cDNA under the control of a suitablepromoter, or by activation of the human glycoprotein α-subunit genethrough the methods described herein.

[0051] By way of example, a sequence coding for a glycoprotein α-subunitcan be included in the DNA construct. This coding sequence is placedunder the transcriptional control of a regulatory sequence that has anucleotide composition that may be identical to or different from thatof the regulatory sequence that is to direct expression of theendogenous FSHβ gene. FIGS. 5-7 illustrate examples of such constructs.

The DNA Construct

[0052] The DNA construct of the invention includes at least a targetingsequence and a regulatory sequence. It may additionally contain an exon;or an exon and an unpaired splice-donor site; or an exon, splice donorsite, intron, and splice acceptor site. The exon, if present, is 3′ ofthe regulatory sequence, and the unpaired splice-donor site is at the 3′end of the exon. The intron and splice acceptor site, if present, are 3′of the splice donor site. In addition, there can be multiple exons andintrons (with appropriate splice donor and acceptor sites) preceding(i.e., 5′ to) the exon flanked by the unpaired splice-donor site. TheDNA in the construct is referred to as exogenous, since the DNA is notan original part of the genome of a host cell. Exogenous DNA may possesssequences identical to or different from portions of the endogenousgenomic DNA present in the cell prior to transfection or infection byviral vector. As used herein, “transfection” means introduction ofplasmid into a cell by chemical and physical means such as calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection,microprojectiles, or biolistic-mediated uptake. As used herein“infection” means introduction of viral nucleic acid into a cell byvirus infection. The various elements included in the DNA construct ofthe invention are described in detail below.

[0053] The DNA construct can also include cis-acting or trans-actingviral sequences (e.g., packaging signals), thereby enabling delivery ofthe construct into the nucleus of a cell via infection by a viralvector. Where necessary, the DNA construct can be disengaged fromvarious steps of a virus life cycle, such as integrase-mediatedintegration in retroviruses or episome maintenance. Disengagement can beaccomplished by appropriate deletions or mutations of viral sequences,such as a deletion of the integrase coding region in a retrovirusvector. Additional details regarding the construction and use of viralvectors are found in Robbins et al., Pharmacol. Ther. 80:35-47, 1998;and Gunzburg et al., Mol. Med. Today 1:410-417, 1995, hereinincorporated by reference.

Targeting Sequences

[0054] Targeting sequences permit homologous recombination of a desiredsequence into a selected site in the host genome. Targeting sequencesare homologous to (i.e., able to homologously recombine with) theirrespective target sites in the host genome.

[0055] A circular DNA construct can employ a single targeting sequence,or two or more separate targeting sequences. A linear DNA construct maycontain two or more separate targeting sequences. The target site towhich a given targeting sequence is homologous can reside within an exonand/or intron of the FSHβ gene, upstream of and immediately adjacent tothe FSHβ-coding region, or upstream of and at a distance from theFSHβ-coding region.

[0056] The first of the two targeting sequences in the construct (or theentire targeting sequence, if there is only one targeting sequence inthe construct) is at least in part derived from the newly disclosedgenomic regions upstream of the FSHβ-coding sequences. This targetingsequence contains a portion of SEQ ID NO:1, e.g., at least 20consecutive nucleotides from the sequence corresponding to positions−7,454 to −1,417 (SEQ ID NO:4) or to positions −696 to −155 (SEQ IDNO:5). The second of the two targeting sequences in the construct maytarget a genomic region upstream of the coding sequence (e.g., alsocontain a portion of SEQ ID NO:4 or 5), or target an exon or intron ofthe gene.

[0057] The targeting sequence(s) may additionally include sequencederived from a previously known region of the FSHβ gene, including thosedescribed herein, as well as regions further upstream which arestructurally uncharacterized but can be mapped by one skilled in theart.

[0058] Genomic fragments that can be used as targeting sequences can beidentified by their ability to hybridize to a probe containing all or aportion of SEQ ID NO:4 or 5. Such a probe can be generated by PCR usingprimers derived from SEQ ID NO:1.

The Regulatory Sequence

[0059] The regulatory sequence of the DNA construct can contain one ormore promoters (e.g., a constitutive, tissue-specific, or induciblepromoter), enhancers, scaffold-attachment regions or matrix attachmentsites, negative regulatory elements, transcription factor binding sites,or combinations of these elements.

[0060] The regulatory sequence can be derived from a eukaryotic (e.g.,mammalian) or viral genome. Useful regulatory sequences include, but arenot limited to, those that regulate the expression of SV40 early or lategenes, cytomegalovirus genes, and adenovirus major late genes. They alsoinclude regulatory regions derived from genes encoding mousemetallothionein-I, elongation factor-1α, collagen (e.g., collagen Iα1,collagen Iα2, and collagen IV), actin (e.g., γ-actin), immunoglobulin,HMG-CoA reductase, glyceraldehyde phosphate dehydrogenase,3-phosphoglyceratekinase, collagenase, stromelysin, fibronectin,vimentin, plasminogen activator inhibitor I, thymosin β4, tissueinhibitors of metalloproteinase, ribosomal proteins, majorhistocompatibility complex molecules, and human leukocyte antigens.

[0061] The regulatory sequence preferably contains transcription factorbinding site, such as a TATA Box, CCAAT Box, AP1, Sp1, or a NF-κBbinding site.

Marker Genes

[0062] If desired, the construct can include a sequence encoding adesired polypeptide, operatively linked to its own promoter. An exampleof this would be a selectable marker gene, which can be used tofacilitate the identification of a targeting event. An amplifiablemarker gene can also be used to facilitate selection of cells havingco-amplified flanking DNA sequences. Cells containing amplified copiesof the amplifiable marker gene can be identified by growth in thepresence of an agent that selects for the expression of the amplifiablegene. The activated endogenous gene will typically be amplified intandem with the amplified selectable marker gene. Cells containingmultiple copies of the activated endogenous gene may produce very highlevels of FSHβ and are thus useful for in vitro protein production andgene therapy.

[0063] The selectable and amplifiable marker genes do not have to lieimmediately adjacent to each other. The amplifiable marker gene andselectable marker gene can be the same gene. One or both of the markergenes can be situated in the intron of the DNA construct. Suitableamplifiable marker genes and selectable marker genes are described inU.S. Pat. No. 5,641,670.

The Exogenous Exon

[0064] The DNA construct may further contain an exon, i.e., a DNAsequence that is copied into RNA and is present in a mature mRNAmolecule. The exon in the construct is referred to herein as anexogenous or construct-derived exon. The exogenous exon can benon-coding, like the first exon of the human FSHβ gene, and in fact canoptionally be identical in sequence to the latter exon. Alternatively,the exogenous exon encodes one or more amino acid residues, or partiallyencodes an amino acid residue (i.e., contains one or two nucleotides ofa codon). When the exon contains a coding sequence, the DNA constructshould be designed such that, upon transcription and splicing, thereading frame of the resulting mRNA is in-frame with the coding regionof the target FSHβ gene. That is, the exogenous exon is spliced to anendogenous exon in a manner that does not change the appropriate readingframe of the portion of the mRNA derived from the endogenous exon.

[0065] The inclusion of a coding exon in the DNA construct allows theproduction of a fusion protein that contains both endogenous FSHβprotein sequence and exogenous protein sequence. Such a hybrid proteinmay combine the structural, enzymatic, or ligand- or receptor-bindingproperties from two or more proteins into one polypeptide. For example,the exogenous exon can encode a cell membrane anchor, a signal peptideto improve cellular secretion, a leader sequence, an enzymatic region, aco-factor binding region, or an epitope tag to facilitate purificationof the FSHβ hybrid protein produced from the recombined gene locus.

The Splice-Donor Site

[0066] The exogenous exon is flanked at its 3′ end by a splice-donorsite. A splice-donor site is a sequence which directs the splicing ofone exon of an RNA transcript to the splice-acceptor site of anotherexon of the RNA transcript. Typically, the first exon lies 5′ of thesecond exon, and the splice-donor site located at the 3′ end of thefirst exon is paired with a splice-acceptor site on the 5′ side of thesecond exon. Splice-donor sites have a characteristic consensus sequencerepresented as (A/C)AGGURAGU (where R denotes a purine), with the GU inthe fourth and fifth positions being required (Jackson, Nucleic AcidsResearch 19:3715-3798, 1991). The first three bases of the splice-donorconsensus site are the last three bases of the exon: i.e., they are notspliced out. Splice-donor sites are functionally defined by theirability to effect the appropriate reaction within the mRNA splicingpathway.

[0067] By way of example, the splice-donor site can be placedimmediately adjacent and 3′ to an ATG codon when the presence of one ormore intervening nucleotides is not required for the exogenous exon tobe in-frame with the second exon of the targeted gene. When theexogenous exon encodes one or more amino acids in-frame with the codingsequence of the targeted gene, the splice-donor site may preferably beplaced immediately adjacent to the exogenous coding sequence on its 3′side.

[0068] The splice-donor site flanking the exogenous exon is unpaired inthe construct, i.e., in the construct itself there is no accompanyingsplice-acceptor site downstream of the splice-donor site to which thelatter can be spliced. Following homologous recombination into thetarget site upstream of the FSHβ coding sequence, what was theconstruct's unpaired splice-donor site is functionally paired with anendogenous splice-acceptor site of an endogenous exon of FSHβ.Processing of the transcript produced from the homologously recombinedFSHβ gene results in splicing of the exogenous exon to thesplice-acceptor site of an endogenous exon.

[0069] The construct of the invention can also include a splice-acceptorsite. This site, in conjunction with a splice-donor site, directs thesplicing of one exon to another exon. Splice-acceptor sites have acharacteristic sequence represented as (Y)₁₀NYAG (SEQ ID NO:7), where Ydenotes any pyrimidine and N denotes any nucleotide (Jackson, NucleicAcids Research 19:3715-3798, 1991).

Introns

[0070] The DNA construct may optionally contain an intron. An intron isa sequence of one or more nucleotides lying between a splice-donor siteand a splice-acceptor site, and is removed, by splicing, from aprecursor RNA molecule in the formation of a mature mRNA molecule.

The CAP Site

[0071] The DNA construct can optionally contain a CAP site. A CAP siteis a specific transcription start site which is associated with andutilized by the regulatory region. This CAP site is located at aposition relative to the regulatory sequence in the construct such thatfollowing homologous recombination, the regulatory sequence directssynthesis of a transcript that begins at the CAP site. Alternatively, noCAP site is included in the construct, and the transcriptional apparatuswill locate by default an appropriate site in the targeted gene to beutilized as a CAP site.

Additional DNA Elements

[0072] The construct may additionally contain sequences which affect thestructure or stability of the RNA or protein produced by homologousrecombination. Optionally, the DNA construct can include a bacterialorigin of replication and bacterial antibiotic resistance markers orother selectable markers, which allow for large-scale plasmidpropagation in bacteria or any other suitable cloning/host system.

[0073] All of the above-described elements of the DNA construct areoperatively linked or functionally placed with respect to each other.That is, upon homologous recombination between the construct and thetargeted genomic DNA, the regulatory sequence can direct the productionof a primary RNA transcript which initiates at a CAP site (optionallyincluded in the construct) and includes (i) sequence corresponding tothe exon and splice-donor site of the construct, if they are present,and (ii) sequence lying between that splice-donor site and theendogenous gene's transcription stop site. The latter sequence mayinclude the FSHβ gene's endogenous regulatory region as well assequences neighboring that region that are normally not transcribed. Inan operatively linked configuration, the splice-donor site of thetargeting construct directs a splicing event to a splice-acceptor siteflanking one of the exons of the endogenous FSHβ gene, such that adesired protein can be produced from the fully spliced maturetranscript. The splice-acceptor site can be endogenous, such that thesplicing event is directed to an endogenous exon. In another embodimentwhere the splice-acceptor site is included in the targeting construct,the splicing event removes the exogenous intron introduced by thetargeting construct.

[0074] The order of elements in the DNA construct can vary. Where theconstruct is a circular plasmid or viral vector, the relative order ofelements in the resulting structure can be, for example: a targetingsequence, plasmid DNA (comprised of sequences used for the selectionand/or replication of the targeting plasmid in a microbial or othersuitable host), selectable marker(s), a regulatory sequence, an exon,and an unpaired splice-donor site.

[0075] Where the construct is linear, the order can be, for example: afirst targeting sequence, a selectable marker gene, a regulatorysequence, an exon, a splice-donor site, and a second targeting sequence;or, in the alternative, a first targeting sequence, a regulatorysequence, an exon, a splice-donor site, a selectable marker gene, and asecond targeting sequence. The order of the elements can also be: afirst targeting sequence, a selectable marker, a regulatory sequence, anexon, a splice-donor site, an intron, a splice-acceptor site, optionallyan internal ribosomal entry site, and second targeting sequence.

[0076] Alternatively, the order can be: a first targeting sequence, afirst selectable marker gene, a regulatory sequence, an exon, asplice-donor site, a second targeting sequence, and a second selectablemarker gene; or, a first targeting sequence, a regulatory sequence, anexon, a splice-donor site, a first selectable marker gene, a secondtargeting sequence, and a second selectable marker gene. Recombinationbetween the targeting sequences flanking the first selectable markerwith homologous sequences in the host genome results in the targetedintegration of the first selectable marker, while the second selectablemarker is not integrated. Desired transfected or infected cells arethose that are stably transfected or infected with the first, but notsecond, selectable marker. Such cells can be selected for by growth in amedium containing an agent which selects for expression of the firstmarker and another agent which selects against the second marker.Transfected or infected cells that have improperly integrated thetargeting construct by a mechanism other than homologous recombinationwould be expected to express the second marker gene and will thereby bekilled in the medium.

[0077] A positively selectable marker is sometimes included in theconstruct to allow for the selection of cells containing amplifiedcopies of that marker. In this embodiment, the order of constructcomponents can be, for example: a first targeting sequence, anamplifiable positively selectable marker, a second selectable marker(optional), a regulatory sequence, an exon, a splice-donor site, and asecond targeting DNA sequence.

[0078] The various elements of the construct can be obtained fromnatural sources (e.g., genomic DNA), or can be produced using geneticengineering techniques or synthetic processes. The regulatory region,CAP site, exon, splice-donor site, and optional intron and spliceacceptor site of the construct can be isolated as a complete unit from,e.g., the human elongation factor-1α (Genbank sequence HUMEF1A) gene orthe cytomegalovirus (Genbank sequence HEHCMVP1) immediate early region.These components can also be isolated from separate genes.

Transfection or Infection and Homologous Recombination

[0079] The DNA construct of the invention can be introduced into thecell, such as a primary, secondary, or immortalized cell, as a singleDNA construct, or as separate DNA sequences which become incorporatedinto the chromosomal or nuclear DNA of a transfected or infected cell.The DNA can be introduced as a linear, double-stranded (with or withoutsingle-stranded regions at one or both ends), single-stranded, orcircular molecule. The DNA construct or its RNA equivalent can also beintroduced as a viral nucleic acid.

[0080] When the construct is introduced into host cells in two separateDNA fragments, the two fragments share DNA sequence homology (overlap)at the 3′ end of one fragment and the 5′ end of the other, while onecarries a first targeting sequence and the other carries a secondtargeting sequence. Upon introduction into a cell, the two fragments canundergo homologous recombination to form a single molecule with thefirst and second targeting sequences flanking the region of overlapbetween the two original fragments. The product molecule is then in aform suitable for homologous recombination with the cellular targetsites. More than two fragments can be used, with each of them designedsuch that they will undergo homologous recombination with each other toultimately form a product suitable for homologous recombination with thecellular target sites as described above.

[0081] The DNA construct of the invention, if not containing aselectable marker itself, can be co-transfected or co-infected withanother construct that contains such a marker. A targeting plasmid maybe cleaved with a restriction enzyme at one or more sites to create alinear or gapped molecule prior to transfection or infection. Theresulting free DNA ends increase the frequency of the desired homologousrecombination event. In addition, the free DNA ends may be treated withan exonuclease to create overhanging 5′ or 3′ single-stranded DNA ends(e.g., at least 30 nucleotides in length, and preferably 100-1000nucleotides in length) to increase the frequency of the desiredhomologous recombination event. In this embodiment, homologousrecombination between the targeting sequence and the genomic target willresult in two copies of the targeting sequences, flanking the elementscontained within the introduced plasmid.

[0082] The DNA constructs may be transfected into cells (preferably invitro) by a variety of physical or chemical methods, includingelectroporation, microinjection, microprojectile bombardment, calciumphosphate precipitation, liposome delivery, or polybrene- or DEAEdextran-mediated transfection.

[0083] The transfected or infected cell is maintained under conditionswhich permit homologous recombination, as described in the art (see,e.g., Capecchi, Science 24:1288-1292, 1989). By “transfected cell” ismeant a cell into which (or into an ancestor of which) a DNA moleculehas been introduced by a means other than using a viral vector. By“infected cell” is meant a cell into which (or into an ancestor ofwhich) a DNA or RNA molecule has been introduced using a viral vector.Viruses known to be useful as vectors include adenovirus,adeno-associated virus, Herpes virus, mumps virus, poliovirus,lentivirus, retroviruses, Sindbis virus, and vaccinia viruses such ascanary pox virus. When the homologously recombinant cell is maintainedunder conditions sufficient to permit transcription of the DNA, theregulatory region introduced by the DNA construct will altertranscription of the FSHβ gene.

[0084] Homologously recombinant cells (i.e., cells that have undergonethe desired homologous recombination) can be identified by phenotypicscreening or by analyzing the culture supernatant in enzyme-linkedimmunosorbent assays (ELISA) for FSHβ. Commercial ELISA kits fordetecting FSHβ are available from Accurate Chemical and Scientific(Westbury, N.Y.). Homologously recombinant cells can also be identifiedby Southern and Northern analyses or by polymerase chain reaction (PCR)screening.

[0085] As used herein, the term “primary cells” includes (i) cellspresent in a suspension of cells isolated from a vertebrate tissuesource (prior to their being plated, i.e., attached to a tissue culturesubstrate such as a dish or flask), (ii) cells present in an explantderived from tissue, (iii) cells plated for the first time, and (iv)cell suspensions derived from these plated cells. Primary cells can alsobe cells as they naturally occur within a human or an animal.

[0086] Secondary cells are cells at all subsequent steps in culturing.That is, the first time that plated primary cells are removed from theculture substrate and replated (passaged), they are referred to hereinas secondary cells, as are all cells in subsequent passages. Secondarycell strains consist of secondary cells which have been passaged one ormore times. Secondary cells typically exhibit a finite number of meanpopulation doublings in culture and the property of contact-inhibited,anchorage-dependent growth (anchorage-dependence does not apply to cellsthat are propagated in suspension culture). Primary and secondary cellsare not immortalized.

[0087] Immortalized cells are cell lines (as opposed to cell strains,with the designation “strain” reserved for primary and secondary cells)that exhibit an apparently unlimited lifespan in culture.

[0088] Cells selected for transfection or infection can fall into fourtypes or categories: (i) cells which do not, as obtained, make orcontain more than trace amounts of the FSHβ protein, (ii) cells whichmake or contain the protein but in quantities other than those desired(such as, in quantities less than the level which is physiologicallynormal for the type of cells as obtained), (iii) cells which make theprotein at a level which is physiologically normal for the type of cellsas obtained, but are to be augmented or enhanced in their content orproduction, and (iv) cells in which it is desirable to change thepattern of regulation or induction of a gene encoding the protein.

[0089] Primary, secondary and immortalized cells to be transfected orinfected by the present method can be obtained from a variety of tissuesand include all appropriate cell types which can be maintained inculture. For example, suitable primary and secondary cells includefibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelialcells, intestinal epithelial cells), endothelial cells, glial cells,neural cells, formed elements of the blood (e.g., lymphocytes, bonemarrow cells), muscle cells, and precursors of these somatic cell types.Where the homologously recombinant cells are to be used in gene therapy,primary cells are preferably obtained from the individual to whom thetransfected or infected primary or secondary cells are to beadministered. However, primary cells can be obtained from a donor (i.e.,an individual other than the recipient) of the same species.

[0090] Examples of immortalized human cell lines useful for proteinproduction or gene therapy include, but are not limited to, 2780ADovarian carcinoma cells (Van der Blick et al., Cancer Res.,48:5927-5932, 1988), A549 (American Type Culture Collection (“ATCC”) CCL185), BeWo (ATCC CCL 98), Bowes Melanoma cells (ATCC CRL 9607), CCRF-CEM(ATCC CCL 119), CCRF-HSB-2 (ATCC CCL 120.1), COLO201 (ATCC CCL 224),COLO205 (ATCC CCL 222), COLO 320DM (ATCC CCL 220), COLO 320HSR (ATCC CCL220.1), Daudi cells (ATCC CCL 213), Detroit 562 (ATCC CCL 138), HeLacells and derivatives of HeLa cells (ATCC CCL 2, 2.1 and 2.2), HCT116(ATCC CCL 247), HL-60 cells (ATCC CCL 240), HT1080 cells (ATCC CCL 121),IMR-32 (ATCC CCL 127), Jurkat cells (ATCC TIB 152), K-562 leukemia cells(ATCC CCL 243), KB carcinoma cells (ATCC CCL 17), KG-1 (ATCC CCL 246),KG-1a (ATCC CCL 246.1), LS123 (ATCC CCL 255), LS174T (ATCC CCL CL-188),LS180 (ATCC CCL CL-187), MCF-7 breast cancer cells (ATCC BTH 22), MOLT-4cells (ATCC CRL 1582), Namalwa cells (ATCC CRL 1432), NCI-H498 (ATCC CCL254), NCI-H508 (ATCC CCL 253), NCI-H548 (ATCC CCL 249), NCI-H716 (ATCCCCL 251), NCI-H747 (ATCC CCL 252), NCI-H1688 (ATCC CCL 257), NCI-H2126(ATCC CCL 256), Raji cells (ATCC CCL 86), RD (ATCC CCL 136), RPMI 2650(ATCC CCL 30), RPMI 8226 cells (ATCC CCL 155), SNU-C2A (ATCC CCL 250.1),SNU-C2B (ATCC CCL 250), SW-13 (ATCC CCL 105), SW48 (ATCC CCL 231), SW403(ATCC CCL 230), SW480 (ATCC CCL 227), SW620 (ATCC CCL 227), SW837 (ATCCCCL 235), SW948 (ATCC CCL 237), SW1116 (ATCC CCL 233), SW1417 (ATCC CCL238), SW1463 (ATCC CCL 234), T84 (ATCC CCL 248), U-937 cells (ATCC CRL1593), WiDr (ATCC CCL 218), and WI-38VA13 subline 2R4 cells (ATCC CLL75.1), as well as heterohybridoma cells produced by fusion of humancells and cells of another species. Secondary human fibroblast strains,such as WI-38 (ATCC CCL 75) and MRC-5 (ATCC CCL 171), may be used. Inaddition, primary, secondary, or immortalized human cells, as well asprimary, secondary, or immortalized cells from other species, can beused for in vitro protein production or gene therapy.

FSHβ-expressing Cells

[0091] Homologously recombinant cells of the invention express FSHβ atdesired levels and are useful for in vitro production of FSHβ and genetherapy.

Protein Production

[0092] Homologously recombinant cells according to this invention can beused for in vitro production of FSHβ. The cells are maintained underconditions, as described in the art, which result in expression ofproteins. The FSHβ protein may be purified from cell lysates or cellsupernatants. A pharmaceutical composition containing the FSHβ proteincan be delivered to a human or an animal by conventional pharmaceuticalroutes known in the art (e.g., oral, intravenous, intramuscular,intranasal, pulmonary, transmucosal, intradermal, transdermal, rectal,intrathecal, subcutaneous, intraperitoneal, or intralesional). Oraladministration may require use of a strategy for protecting the proteinfrom degradation in the gastrointestinal tract: e.g., by encapsulationin polymeric microcapsules.

Gene Therapy

[0093] Homologously recombinant cells of the present invention areuseful as populations of homologously recombinant cell lines, aspopulations of homologously recombinant primary or secondary cells, ashomologously recombinant clonal cell strains or lines, as homologouslyrecombinant heterogenous cell strains or lines, and as cell mixtures inwhich at least one representative cell of one of the four precedingcategories of homologously recombinant cells is present. Such cells maybe used in a delivery system for treating infertility, for enhancingfertility in a human or animal, or for treating any other conditionstreatable with FSHβ.

[0094] Homologously recombinant primary cells, clonal cell strains orheterogenous cell strains are administered to an individual in whom theabnormal or undesirable condition is to be treated or prevented, insufficient quantity and by an appropriate route, to express or makeavailable the protein or exogenous DNA at physiologically relevantlevels. A physiologically relevant level is one which eitherapproximates the level at which the product is normally produced in thebody or results in improvement of the abnormal or undesirable condition.If the cells are syngeneic with respect to a immunocompetent recipient,the cells can be administered or implanted intravenously,intraarterially, subcutaneously, intraperitoneally, intraomentally,subrenal capsularly, intrathecally, intracranially, or intramuscularly.

[0095] If the cells are not syngeneic and the recipient isimmunocompetent, the homologously recombinant cells to be administeredcan be enclosed in one or more semipermeable barrier devices. Thepermeability properties of the device are such that the cells areprevented from leaving the device upon implantation into a subject, butthe therapeutic protein is freely permeable and can leave the barrierdevice and enter the local space surrounding the implant or enter thesystemic circulation. See, e.g., U.S. Pat. Nos. 5,641,670, 5,470,731,5,620,883, 5,487,737, and co-owned U.S. Patent Application entitled“Delivery of Therapeutic Proteins” (inventors: Justin C. Lamsa andDouglas A. Treco), filed Apr. 16, 1999, all herein incorporated byreference. The barrier device can be implanted at any appropriate site:e.g., intraperitoneally, intrathecally, subcutaneously, intramuscularly,within the kidney capsule, or within the omentum.

[0096] Barrier devices are particularly useful and allow homologouslyrecombinant immortalized cells, homologously recombinant cells fromanother species (homologously recombinant xenogeneic cells), or cellsfrom a nonhisto-compatibility-matched donor (homologously recombinantallogeneic cells) to be implanted for treatment of a subject. Thedevices retain cells in a fixed position in vivo, while protecting thecells from the host's immune system. Barrier devices also allowconvenient short-term (i.e., transient) therapy by allowing readyremoval of the cells when the treatment regimen is to be halted for anyreason. Transfected or infected xenogeneic and allogeneic cells may alsobe used in the absence of barrier devices for short-term gene therapy.In that case, the FSHβ produced by the cells will be delivered in vivountil the cells are rejected by the host's immune system.

[0097] A number of synthetic, semisynthetic, or natural filtrationmembranes can be used for this purpose, including, but not limited to,cellulose, cellulose acetate, nitrocellulose, polysulfone,polyvinylidene difluoride, polyvinyl chloride polymers and polymers ofpolyvinyl chloride derivatives. Barrier devices can be utilized to allowprimary, secondary, or immortalized cells from another species to beused for gene therapy in humans.

[0098] Another type of device useful in the gene therapy of theinvention is an implantable collagen matrix in which the cells areembedded. Such a device, which can contain beads to which the cellsattach, is described in WO 97/15195, herein incorporated by reference.

[0099] The number of cells needed for a given dose or implantationdepends on several factors, including the expression level of theprotein, the size and condition of the host animal, and the limitationsassociated with the implantation procedure. Usually the number of cellsimplanted in an adult human or other similarly-sized animal is in therange of 1×10⁴ to 5×10¹⁰, and preferably 1×10⁸ to 1×10⁹. If desired,they may be implanted at multiple sites in the patient, either at onetime or over a period of months or years. The dosage may be repeated asneeded.

Other Embodiments

[0100] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not to limit thescope of the invention, which is defined by the scope of the appendedclaims.

[0101] Other aspects, advantages, and modifications are within the scopeof the following claims.

1 7 1 7622 DNA Homo sapiens 1 ggatccgaga acatagaagg agcaggtaatttatcaaggc atgaacacgg gtgcttaatt 60 tcctattttg aggccaggca tggtggctcacacctgtaat cccaacactt taggaagcca 120 aggtgggtgg attgcttgag tctaggattttgagaccagc ctggccaaca tggcgaaatc 180 ctgtctctac taaaaatact aaaattaaccagtcatggtg gtggtgtgcc tttagtccca 240 gctactctgg tggctgaggc acaagaatcacttgaacctg ggaggcagag gttgcagtga 300 gctgagactg tgccacttca ctccagcctgggtgacagag taagattctg tctcaaaaaa 360 tatgtatata tacacacata taatagatacataaacatat atacatatat aatatataaa 420 tatatatatt atatataata tataaacatatataaatata tatatatata tatatatata 480 tatataaacc aaacataaag gaataattttgggggaaaat cttcataaat gaaagaacaa 540 cataggctgt tgagtatatg cacagaaattcaagagatct tccagcaatt gaagacattg 600 gtttaccaga attcacaaaa gaagtcagctgtgcatttaa agtagaatgt gatgagtgtt 660 accactgagg taggaactgg gaactaaggaagcgtaagac agaaagtgct gaactgagag 720 ttgggcattg gaggctgtgt aaggcagggtaagtgaatgt ctcctagaag ctacctttaa 780 atggagtttt gaagtacttg taggagtagcttaggtgaaa agaagaggag aaacatgtat 840 caggcagagg gactagaacc ttattaccttcaaagaagaa gcaaaaagaa tacatgtgac 900 tttgaggtgg tgggaggtgc tttaagccaatataggtgaa tttgacatag gacttcccta 960 aataatgttc ggtcatttgt taaatattgagtgatatatc actgtattaa agcccaagag 1020 ttgcttttat atagaaagaa gaaaaaagcccaagagagtt ttatttctag agggaatatt 1080 ttctagaaat aaaggaaggt gtatcagccagtttctagtc aggaaaacag aaatcacacc 1140 tgatatgcaa aatagaggaa aatcagggaattcattaatc cagagatttg gttgctcaag 1200 tattagattg ctgaaaagcc agacagggaatatgaggcaa tcagagataa gtattagtga 1260 caagctccat ttatgtgcag gattggagggacataggtgg ggttcccaga agccagaagg 1320 tgagaccacc tagcagaagc tcaaaccacagctggggttt cctcacaaaa gctgggacca 1380 ccaggaggag ctgtccaatg ggatctggagccagggagat catgcagtca ctaccaggaa 1440 gggaagcaga atgtaaaagg tagagagaaatactccaact gcttccttgc attcactttc 1500 caatctccat tcacaaaggc aaaaacctgctaatacagca gagtgggaaa agcagcctgc 1560 caaggtcctt tctcccacaa aacagagcacaaaaccaagc aaaaacaagg aatgcatttg 1620 atagcaaaca ggctatggac caacccaacataaaagaaat gatgagtgat ttcttttttc 1680 atttggttca agaaaagtat ttcagtaactattatgtaac agaaattcta tttattttgg 1740 ggaattcaaa ggtgaataaa aaagaactctaaatttttat caataaaata tttcaaaaac 1800 ctcaatgaga gtaatggcat taactagcaaatatgctaat gagatgagct agccataaga 1860 ggcttagaat tgagagaaag gtctgggggcctcttgacag gccaaattca gagctgtttg 1920 tgggaatctc tgacctaact gcaggtggaaatataaatat gggcatttag aatagtggcc 1980 caaactttgg atgatttctg tcttggggtctctccaatta atgggattga tgagaactgt 2040 agaccactga ggtcaccatg gctcaatgaatagtcccctg gctttggagt caaactgacc 2100 tgaatatgaa ccccagcttt gctacttacaggttgcattt atcctcagtt ttctcatctt 2160 tcaaagaaga acagtaactt ctttaaaaggttattgtagg ctgggtgcag tggctcacgc 2220 ctgtaatcgc agcactttgg gaggcggaggctagtggatc acttgaggcc aggagttgga 2280 aactagcctg gccaacatgg tgaaactctgtctctacaaa aagaaattta aaaaattttg 2340 ctgggtgtgg tggcacacac ctggaattccagctacctgg gaggccgagg catgagcatc 2400 acttgagtct ggaaagcaga gggttgcagtgagccaagat tgtaccactg tactcaagcc 2460 tgggtgacac agtgagacct tgtctaaaaaaaaaaaaggt tattgtgtta ttgtaaatat 2520 tgtatatgaa cttctattta acatgtttagttaaatgcct gtgtaattgt ccaatgtgct 2580 cttctagctc actgcacaga caaaactgattcactgaaat catggaattg cagcaaagaa 2640 caaatctaat taatgtaggt caaacgggaggactggagtt attattcaaa tcagtctccc 2700 tgaaaactca gaggctaggg ttttatggataatttggtgg gcaggggact agggaatggg 2760 tgctgctgat tggttgggga atgaaatagtaagattgtgg aaaactgtcc tccttcattg 2820 agtctgcttc cgggtgtagg ccacacgaccagttgagtca tgaagcatgc gtccaagtgg 2880 agtcagtttg ttgccagaat gcaaaagcctgaaaaatgtc tcaaatgatc aactgtaggc 2940 tccacaataa tgatattatc tataggagcaattggggaag taacaaatct tgtgacctct 3000 ggacacataa ctcctgaact agtaagggattataaaaacc atgcctatat cttatcagaa 3060 ttcaggtccc cccataatcc taatctcacagcatttcatt tgtttagaaa ggccattttc 3120 agtccctgag caaggagggg gttagttttaggataggact attatccttg cttcgttaaa 3180 ctataaacta aattcctccc atggttagcttggcctacac ctaagaatga gtgagaacag 3240 ccagcctgtg aggctagagg caagatggagtcagccatgc tagatttatc tcactgtcat 3300 aacctttgca aaggcagttt cacctgggacataggaggta ctcaatgaaa aagaagctat 3360 taatattaaa attttaaaaa tgaatttaaggaactaatac tatgtacata ttagtcatta 3420 aaacaaagtg gttcatttac attcacacaaataaatcttg tgattataca taggtaatat 3480 gaaaaacttt gttttctttc ataatacaaggtattagcaa tagatatagt aatgttagca 3540 ttcctttgga aaaaatgaaa agatttataattttccaaga atcattagta tttttattta 3600 atatacataa tataaaattt attcattctataacttggaa atatgcttgc ttaccaatta 3660 ctgacagatt tcaaaatatt tctatactcacaatattcat ttacataaat attgatttgg 3720 tacttacaat gtgtactgct atgctaagttttgtctttgt caaacatatt ttataaaatc 3780 ataatcctag atgaatccaa cttttggtaacccacgtgcc tgaacccctg ctgttaacag 3840 gcaaagtgtg gtaggtacag atctatacctaccaccttcc tctacccacc agcatctgca 3900 cccaccaccc ctccccaccc accattatctataccaacca cccctcccaa cctaccagca 3960 tctgcaccca ccacaccgcc cacccaccaccatgtacact cactacacct tccagccatc 4020 accatctgca cccatcactc ctccccatccacaagcatct gcacccacca catttcccta 4080 cctaccagca tcttcactca ccacctctccacccaccagc atctgcaccc acaacccctc 4140 ctcacccacc agagtctgca tccatcacacttgcccactc gctagcatct gcaccatcaa 4200 gctctgcctt cttgcctaat acgggatgagctctccatgg ttctgcctaa agacaatgct 4260 tccactcctc ttctataacc catttccttttacctcttca agtacacttc agaacttctc 4320 tctccttctg ataccaactt tttccactttactcaatcat tcctatcacc atacaaacgt 4380 gtttatttct cccatcttaa agttaaaaatcaaaagaaaa ttgtctgcgg ccaggcacgg 4440 tggctcacgc ctgtaatccc aacactttgggaggccaagg agggttggat gacttaaggt 4500 taggagttca agaccagcct ggccaacatggtgaaaccca tctctactaa aaatacaaaa 4560 attagccagg catggtggca catgcctgtagtctcaggta cttgggaggc tgaggccaga 4620 gaatggcttg aacccgggag gcagaggttgcagtgagccg agattgtgcc cttgcactcc 4680 agcctgggtg acagagtgag actccatctcaaaaataaaa aataaaaata aaacaaaaga 4740 aagttatttt tacccaacat ccacattaaccaaataccca tttctttatt gatctttgta 4800 aaaaaaagct cttggaaaaa ttgtctatattcactatgac ttatctcctc caaatcactt 4860 aaacacatac caatcaggtt tttgttttcatcattccaaa gtaactttta cagccaagga 4920 cagtagcgaa ctttacatcg catatgcattgtgaagttct tgatcctcat cttacttaac 4980 ctgtcagcag tatctgacac aggtgtcactggctcctccc tgagatgctc tctttatttg 5040 gctttgggga caccatattc tccccattcctactttcctc aatggccctc ctcagtctcc 5100 tttggaaaga ggaaaaagaa acttcattatctcctggatg tagtacaaac aactcaagct 5160 caacatgtgc atactgaact ccatttccttttcccaaact tcgacattta cagccatccc 5220 ctttcagctg atagcaagtt tatccttccagctactcaaa ccagaatctt tagagccatc 5280 cttgaccctt ttcctcctct cacactcaacatctatccat cagaaaattt tgttggttct 5340 actttcaaaa tgcatacaga gtcagagcatgtctcattac ctccaatagc taccatacta 5400 gtctgaacaa acatcatttc tcacctgggttattgaacaa acatcatttc tcacctgggt 5460 tattgatagc atcctaacgg gtcttcctgtttcttggttc ccctatatta gcaacacagc 5520 agtcagagga gtccttttag aactcaatcagatcatgtca cgtcactcct ctacttaaaa 5580 tccttcaatg ggtcccatta cacaaagagtacaaaccaga gcccttacac tggtctacaa 5640 gttccaacat ttgactcctg ttatctctctgacatcatat tctaatatta ctgctgttgt 5700 ccttttgctc cagtcacact gtttgattagtaaatattta ttaaacaaag caatcctagt 5760 ctccaaagag atcatagttt attggaggaaacaagagcct ataaatggtt acacacagaa 5820 ggtagtgatt atggttctcc ctcacctcccatcctaaact ttgacaggtg aaactcccct 5880 ggatgttgaa ggttgaggaa tttgccagggttcagggtgg tgttggagga ggcagggagg 5940 aagcaaggac atttcaggca ggaagaacattacatgcaaa gatctaaaga tatgaatcag 6000 caacatattt atggaattac aagtaaagtagaaagttctt gctaaaacat caaaaaataa 6060 agatttgtga ttagggggcc agaatgtgggagggaaagag agatacagtt cacactttta 6120 gacaggagcc agatcatgaa atgttttctctttgtttgtt tcttccttca cagcttttga 6180 tatgctcttg gagcaattta ttaaccatattttttaatgc atctcctgaa cagagtcaaa 6240 gcaatacttg gaaaggactc tgaatttcctgatttaaaga tacaaaagaa aaatctggag 6300 tcacaattaa tttgagaagg taaaggagtgggtgtgctac tgtatcaaat ttaatttgta 6360 caaaatcatc atctctagta acattattttttctaatcta ctgcgtttag actactttag 6420 taaagcttga tctccctgtc tatctaaacactgattcact tacagcaagc ttcaggctag 6480 cattggtcat attaataccc aacaaatccacaaggtgtta gttgcacatg attttgtata 6540 aaaggtgaac tgagatttca ttcagtctacagctcttgcc aggcaaggca gccgaccaca 6600 ggtgagtctt ggcatctacc gttttcaagtgtgacagcta cttttgaaat tacagatttg 6660 tcaggacatg gaggacaaaa ctagagcttctcactactgt tgtgtaggaa atttatgctt 6720 gtcaacctgg cttgtaaaat atggttaatataacgtaatc actgttagca agtaactgac 6780 tttatagacc aatatgcctc tcttctgaaatggtcttatt ttaaacaaat gtgagcaaaa 6840 gaaaatattt atgagattct aaaaatgaagacataatttt gtagtataga attttcttgg 6900 ccaggaatgg tggctcatgc ttgtaatcccagcactttgg gaggccaagg tcagaggatt 6960 gcttgagcct ggaaggttga agatgcagtgattcatgatt ataccactgc actccagcct 7020 gggcaacaga gcaagaccct gtctcaagaaaagaaaagaa ttttattttt cttttcagac 7080 aaaaatagac tttaaaataa taatggaagaacaaatatga tgatcacaat tatcagagta 7140 attactttat gacagtcagc aataagattctaatctttaa atattcctct gcttaaatca 7200 ttatattgga gttttgatct ataatatattcccaccctga cccaaaaatt gaagaaggac 7260 aaggaaaaat gttgttccaa gaaacaaagatgtaagtaaa aaggcataag gaaggaaaaa 7320 aaacttttga agcaaaatgt gattgaggaggatgagcaga ccaattattt ttggtttggt 7380 cagcttacat aatgattatc gttctttggtttctcagttt ctagtgggct tcattgtttg 7440 cttcccagac caggatgaag acactccagtttttcttcct tttctgttgc tggaaagcaa 7500 tctgctgcaa tagctgtgag ctgaccaacatcaccattgc aatagagaaa gaagaatgtc 7560 gtttctgcat aagcatcaac accacttggtgtgctggcta ctgctacacc agggtaggta 7620 cc 7622 2 53 PRT Homo sapiens 2Met Lys Thr Leu Gln Phe Phe Phe Leu Phe Cys Cys Trp Lys Ala Ile 1 5 1015 Cys Cys Asn Ser Cys Glu Leu Thr Asn Ile Thr Ile Ala Ile Glu Lys 20 2530 Glu Glu Cys Arg Phe Cys Ile Ser Ile Asn Thr Thr Trp Cys Ala Gly 35 4045 Tyr Cys Tyr Thr Arg 50 3 40 DNA Homo sapiens 3 ttggcatcta ccgttttcaagtggtgacag ctacttttga 40 4 6038 DNA Homo sapiens 4 ggatccgaga acatagaaggagcaggtaat ttatcaaggc atgaacacgg gtgcttaatt 60 tcctattttg aggccaggcatggtggctca cacctgtaat cccaacactt taggaagcca 120 aggtgggtgg attgcttgagtctaggattt tgagaccagc ctggccaaca tggcgaaatc 180 ctgtctctac taaaaatactaaaattaacc agtcatggtg gtggtgtgcc tttagtccca 240 gctactctgg tggctgaggcacaagaatca cttgaacctg ggaggcagag gttgcagtga 300 gctgagactg tgccacttcactccagcctg ggtgacagag taagattctg tctcaaaaaa 360 tatgtatata tacacacatataatagatac ataaacatat atacatatat aatatataaa 420 tatatatatt atatataatatataaacata tataaatata tatatatata tatatatata 480 tatataaacc aaacataaaggaataatttt gggggaaaat cttcataaat gaaagaacaa 540 cataggctgt tgagtatatgcacagaaatt caagagatct tccagcaatt gaagacattg 600 gtttaccaga attcacaaaagaagtcagct gtgcatttaa agtagaatgt gatgagtgtt 660 accactgagg taggaactgggaactaagga agcgtaagac agaaagtgct gaactgagag 720 ttgggcattg gaggctgtgtaaggcagggt aagtgaatgt ctcctagaag ctacctttaa 780 atggagtttt gaagtacttgtaggagtagc ttaggtgaaa agaagaggag aaacatgtat 840 caggcagagg gactagaaccttattacctt caaagaagaa gcaaaaagaa tacatgtgac 900 tttgaggtgg tgggaggtgctttaagccaa tataggtgaa tttgacatag gacttcccta 960 aataatgttc ggtcatttgttaaatattga gtgatatatc actgtattaa agcccaagag 1020 ttgcttttat atagaaagaagaaaaaagcc caagagagtt ttatttctag agggaatatt 1080 ttctagaaat aaaggaaggtgtatcagcca gtttctagtc aggaaaacag aaatcacacc 1140 tgatatgcaa aatagaggaaaatcagggaa ttcattaatc cagagatttg gttgctcaag 1200 tattagattg ctgaaaagccagacagggaa tatgaggcaa tcagagataa gtattagtga 1260 caagctccat ttatgtgcaggattggaggg acataggtgg ggttcccaga agccagaagg 1320 tgagaccacc tagcagaagctcaaaccaca gctggggttt cctcacaaaa gctgggacca 1380 ccaggaggag ctgtccaatgggatctggag ccagggagat catgcagtca ctaccaggaa 1440 gggaagcaga atgtaaaaggtagagagaaa tactccaact gcttccttgc attcactttc 1500 caatctccat tcacaaaggcaaaaacctgc taatacagca gagtgggaaa agcagcctgc 1560 caaggtcctt tctcccacaaaacagagcac aaaaccaagc aaaaacaagg aatgcatttg 1620 atagcaaaca ggctatggaccaacccaaca taaaagaaat gatgagtgat ttcttttttc 1680 atttggttca agaaaagtatttcagtaact attatgtaac agaaattcta tttattttgg 1740 ggaattcaaa ggtgaataaaaaagaactct aaatttttat caataaaata tttcaaaaac 1800 ctcaatgaga gtaatggcattaactagcaa atatgctaat gagatgagct agccataaga 1860 ggcttagaat tgagagaaaggtctgggggc ctcttgacag gccaaattca gagctgtttg 1920 tgggaatctc tgacctaactgcaggtggaa atataaatat gggcatttag aatagtggcc 1980 caaactttgg atgatttctgtcttggggtc tctccaatta atgggattga tgagaactgt 2040 agaccactga ggtcaccatggctcaatgaa tagtcccctg gctttggagt caaactgacc 2100 tgaatatgaa ccccagctttgctacttaca ggttgcattt atcctcagtt ttctcatctt 2160 tcaaagaaga acagtaacttctttaaaagg ttattgtagg ctgggtgcag tggctcacgc 2220 ctgtaatcgc agcactttgggaggcggagg ctagtggatc acttgaggcc aggagttgga 2280 aactagcctg gccaacatggtgaaactctg tctctacaaa aagaaattta aaaaattttg 2340 ctgggtgtgg tggcacacacctggaattcc agctacctgg gaggccgagg catgagcatc 2400 acttgagtct ggaaagcagagggttgcagt gagccaagat tgtaccactg tactcaagcc 2460 tgggtgacac agtgagaccttgtctaaaaa aaaaaaaggt tattgtgtta ttgtaaatat 2520 tgtatatgaa cttctatttaacatgtttag ttaaatgcct gtgtaattgt ccaatgtgct 2580 cttctagctc actgcacagacaaaactgat tcactgaaat catggaattg cagcaaagaa 2640 caaatctaat taatgtaggtcaaacgggag gactggagtt attattcaaa tcagtctccc 2700 tgaaaactca gaggctagggttttatggat aatttggtgg gcaggggact agggaatggg 2760 tgctgctgat tggttggggaatgaaatagt aagattgtgg aaaactgtcc tccttcattg 2820 agtctgcttc cgggtgtaggccacacgacc agttgagtca tgaagcatgc gtccaagtgg 2880 agtcagtttg ttgccagaatgcaaaagcct gaaaaatgtc tcaaatgatc aactgtaggc 2940 tccacaataa tgatattatctataggagca attggggaag taacaaatct tgtgacctct 3000 ggacacataa ctcctgaactagtaagggat tataaaaacc atgcctatat cttatcagaa 3060 ttcaggtccc cccataatcctaatctcaca gcatttcatt tgtttagaaa ggccattttc 3120 agtccctgag caaggagggggttagtttta ggataggact attatccttg cttcgttaaa 3180 ctataaacta aattcctcccatggttagct tggcctacac ctaagaatga gtgagaacag 3240 ccagcctgtg aggctagaggcaagatggag tcagccatgc tagatttatc tcactgtcat 3300 aacctttgca aaggcagtttcacctgggac ataggaggta ctcaatgaaa aagaagctat 3360 taatattaaa attttaaaaatgaatttaag gaactaatac tatgtacata ttagtcatta 3420 aaacaaagtg gttcatttacattcacacaa ataaatcttg tgattataca taggtaatat 3480 gaaaaacttt gttttctttcataatacaag gtattagcaa tagatatagt aatgttagca 3540 ttcctttgga aaaaatgaaaagatttataa ttttccaaga atcattagta tttttattta 3600 atatacataa tataaaatttattcattcta taacttggaa atatgcttgc ttaccaatta 3660 ctgacagatt tcaaaatatttctatactca caatattcat ttacataaat attgatttgg 3720 tacttacaat gtgtactgctatgctaagtt ttgtctttgt caaacatatt ttataaaatc 3780 ataatcctag atgaatccaacttttggtaa cccacgtgcc tgaacccctg ctgttaacag 3840 gcaaagtgtg gtaggtacagatctatacct accaccttcc tctacccacc agcatctgca 3900 cccaccaccc ctccccacccaccattatct ataccaacca cccctcccaa cctaccagca 3960 tctgcaccca ccacaccgcccacccaccac catgtacact cactacacct tccagccatc 4020 accatctgca cccatcactcctccccatcc acaagcatct gcacccacca catttcccta 4080 cctaccagca tcttcactcaccacctctcc acccaccagc atctgcaccc acaacccctc 4140 ctcacccacc agagtctgcatccatcacac ttgcccactc gctagcatct gcaccatcaa 4200 gctctgcctt cttgcctaatacgggatgag ctctccatgg ttctgcctaa agacaatgct 4260 tccactcctc ttctataacccatttccttt tacctcttca agtacacttc agaacttctc 4320 tctccttctg ataccaactttttccacttt actcaatcat tcctatcacc atacaaacgt 4380 gtttatttct cccatcttaaagttaaaaat caaaagaaaa ttgtctgcgg ccaggcacgg 4440 tggctcacgc ctgtaatcccaacactttgg gaggccaagg agggttggat gacttaaggt 4500 taggagttca agaccagcctggccaacatg gtgaaaccca tctctactaa aaatacaaaa 4560 attagccagg catggtggcacatgcctgta gtctcaggta cttgggaggc tgaggccaga 4620 gaatggcttg aacccgggaggcagaggttg cagtgagccg agattgtgcc cttgcactcc 4680 agcctgggtg acagagtgagactccatctc aaaaataaaa aataaaaata aaacaaaaga 4740 aagttatttt tacccaacatccacattaac caaataccca tttctttatt gatctttgta 4800 aaaaaaagct cttggaaaaattgtctatat tcactatgac ttatctcctc caaatcactt 4860 aaacacatac caatcaggtttttgttttca tcattccaaa gtaactttta cagccaagga 4920 cagtagcgaa ctttacatcgcatatgcatt gtgaagttct tgatcctcat cttacttaac 4980 ctgtcagcag tatctgacacaggtgtcact ggctcctccc tgagatgctc tctttatttg 5040 gctttgggga caccatattctccccattcc tactttcctc aatggccctc ctcagtctcc 5100 tttggaaaga ggaaaaagaaacttcattat ctcctggatg tagtacaaac aactcaagct 5160 caacatgtgc atactgaactccatttcctt ttcccaaact tcgacattta cagccatccc 5220 ctttcagctg atagcaagtttatccttcca gctactcaaa ccagaatctt tagagccatc 5280 cttgaccctt ttcctcctctcacactcaac atctatccat cagaaaattt tgttggttct 5340 actttcaaaa tgcatacagagtcagagcat gtctcattac ctccaatagc taccatacta 5400 gtctgaacaa acatcatttctcacctgggt tattgaacaa acatcatttc tcacctgggt 5460 tattgatagc atcctaacgggtcttcctgt ttcttggttc ccctatatta gcaacacagc 5520 agtcagagga gtccttttagaactcaatca gatcatgtca cgtcactcct ctacttaaaa 5580 tccttcaatg ggtcccattacacaaagagt acaaaccaga gcccttacac tggtctacaa 5640 gttccaacat ttgactcctgttatctctct gacatcatat tctaatatta ctgctgttgt 5700 ccttttgctc cagtcacactgtttgattag taaatattta ttaaacaaag caatcctagt 5760 ctccaaagag atcatagtttattggaggaa acaagagcct ataaatggtt acacacagaa 5820 ggtagtgatt atggttctccctcacctccc atcctaaact ttgacaggtg aaactcccct 5880 ggatgttgaa ggttgaggaatttgccaggg ttcagggtgg tgttggagga ggcagggagg 5940 aagcaaggac atttcaggcaggaagaacat tacatgcaaa gatctaaaga tatgaatcag 6000 caacatattt atggaattacaagtaaagta gaaagttc 6038 5 542 DNA Homo sapiens 5 tcactgttag caagtaactgactttataga ccaatatgcc tctcttctga aatggtctta 60 ttttaaacaa atgtgagcaaaagaaaatat ttatgagatt ctaaaaatga agacataatt 120 ttgtagtata gaattttcttggccaggaat ggtggctcat gcttgtaatc ccagcacttt 180 gggaggccaa ggtcagaggattgcttgagc ctggaaggtt gaagatgcag tgattcatga 240 ttataccact gcactccagcctgggcaaca gagcaagacc ctgtctcaag aaaagaaaag 300 aattttattt ttcttttcagacaaaaatag actttaaaat aataatggaa gaacaaatat 360 gatgatcaca attatcagagtaattacttt atgacagtca gcaataagat tctaatcttt 420 aaatattcct ctgcttaaatcattatattg gagttttgat ctataatata ttcccaccct 480 gacccaaaaa ttgaagaaggacaaggaaaa atgttgttcc aagaaacaaa gatgtaagta 540 aa 542 6 2125 DNA Homosapiens 6 gatctatacc taccaccttc ctctacccac cagcatctgc acccaccacccctccccacc 60 caccattatc tataccaacc acccctccca acctaccagc atctgcacccaccacaccgc 120 ccacccacca ccatgtacac tcactacacc ttccagccat caccatctgcacccatcact 180 cctccccatc cacaagcatc tgcacccacc acatttccct acctaccagcatcttcactc 240 accacctctc cacccaccag catctgcacc cacaacccct cctcacccaccagagtctgc 300 atccatcaca cttgcccact cgctagcatc tgcaccatca agctctgccttcttgcctaa 360 tacgggatga gctctccatg gttctgccta aagacaatgc ttccactcctcttctataac 420 ccatttcctt ttacctcttc aagtacactt cagaacttct ctctccttctgataccaact 480 ttttccactt tactcaatca ttcctatcac catacaaacg tgtttatttctcccatctta 540 aagttaaaaa tcaaaagaaa attgtctgcg gccaggcacg gtggctcacgcctgtaatcc 600 caacactttg ggaggccaag gagggttgga tgacttaagg ttaggagttcaagaccagcc 660 tggccaacat ggtgaaaccc atctctacta aaaatacaaa aattagccaggcatggtggc 720 acatgcctgt agtctcaggt acttgggagg ctgaggccag agaatggcttgaacccggga 780 ggcagaggtt gcagtgagcc gagattgtgc ccttgcactc cagcctgggtgacagagtga 840 gactccatct caaaaataaa aaataaaaat aaaacaaaag aaagttatttttacccaaca 900 tccacattaa ccaaataccc atttctttat tgatctttgt aaaaaaaagctcttggaaaa 960 attgtctata ttcactatga cttatctcct ccaaatcact taaacacataccaatcaggt 1020 ttttgttttc atcattccaa agtaactttt acagccaagg acagtagcgaactttacatc 1080 gcatatgcat tgtgaagttc ttgatcctca tcttacttaa cctgtcagcagtatctgaca 1140 caggtgtcac tggctcctcc ctgagatgct ctctttattt ggctttggggacaccatatt 1200 ctccccattc ctactttcct caatggccct cctcagtctc ctttggaaagaggaaaaaga 1260 aacttcatta tctcctggat gtagtacaaa caactcaagc tcaacatgtgcatactgaac 1320 tccatttcct tttcccaaac ttcgacattt acagccatcc cctttcagctgatagcaagt 1380 ttatccttcc agctactcaa accagaatct ttagagccat ccttgacccttttcctcctc 1440 tcacactcaa catctatcca tcagaaaatt ttgttggttc tactttcaaaatgcatacag 1500 agtcagagca tgtctcatta cctccaatag ctaccatact agtctgaacaaacatcattt 1560 ctcacctggg ttattgaaca aacatcattt ctcacctggg ttattgatagcatcctaacg 1620 ggtcttcctg tttcttggtt cccctatatt agcaacacag cagtcagaggagtcctttta 1680 gaactcaatc agatcatgtc acgtcactcc tctacttaaa atccttcaatgggtcccatt 1740 acacaaagag tacaaaccag agcccttaca ctggtctaca agttccaacatttgactcct 1800 gttatctctc tgacatcata ttctaatatt actgctgttg tccttttgctccagtcacac 1860 tgtttgatta gtaaatattt attaaacaaa gcaatcctag tctccaaagagatcatagtt 1920 tattggagga aacaagagcc tataaatggt tacacacaga aggtagtgattatggttctc 1980 cctcacctcc catcctaaac tttgacaggt gaaactcccc tggatgttgaaggttgagga 2040 atttgccagg gttcagggtg gtgttggagg aggcagggag gaagcaaggacatttcaggc 2100 aggaagaaca ttacatgcaa agatc 2125 7 14 DNA Homo sapiensmisc_feature (1)...(14) n = A,T,C or G 7 yyyyyyyyyy nyag 14

What is claimed is:
 1. A DNA construct that alters expression of anendogenous FSHβ gene in a mammalian cell upon integration into thegenome of the cell via homologous recombination, the constructcomprising a targeting sequence containing at least 20 contiguousnucleotides from SEQ ID NO:4 or 5 and a transcriptional regulatorysequence.
 2. The DNA construct of claim 1 , wherein the constructfurther comprises an exon and a splice-donor site.
 3. The DNA constructof claim 2 , wherein the construct further comprises, downstream fromthe splice-donor site, an intron and a splice-acceptor site.
 4. The DNAconstruct of claim 1 , wherein the construct further comprises aselectable marker gene.
 5. The DNA construct of claim 1 , wherein thetarget sequence contains at least 50 contiguous nucleotides from SEQ IDNO:4 or
 5. 6. An isolated nucleic acid comprising at least 20 contiguousnucleotides of SEQ ID NO:4 or its complement.
 7. The isolated nucleicacid of claim 6 , wherein the isolated nucleic acid comprises at least50 contiguous nucleotides of SEQ ID NO:4 or its complement.
 8. Theisolated nucleic acid of claim 6 , wherein the isolated nucleic acidcomprises at least 100 contiguous nucleotides of SEQ ID NO:4 or itscomplement.
 9. The isolated nucleic acid of claim 6 , wherein theisolated nucleic acid comprises at least 200 contiguous nucleotides ofSEQ ID NO:4 or its complement.
 10. The isolated nucleic acid of claim 6, wherein the isolated nucleic acid comprises at least 500 contiguousnucleotides of SEQ ID NO:4 or its complement.
 11. The isolated nucleicacid of claim 6 , wherein the isolated nucleic acid comprises at least1000 contiguous nucleotides of SEQ ID NO:4 or its complement.
 12. Theisolated nucleic acid of claim 6 , wherein the isolated nucleic acidcomprises SEQ ID NO:4 or its complement.
 13. An isolated nucleic acidcomprising at least 20 contiguous nucleotides of SEQ ID NO:5 or itscomplement.
 14. The isolated nucleic acid of claim 13 , wherein theisolated nucleic acid comprises at least 50 contiguous nucleotides ofSEQ ID NO:5 or its complement.
 15. The isolated nucleic acid of claim 13, wherein the isolated nucleic acid comprises at least 100 contiguousnucleotides of SEQ ID NO:5 or its complement.
 16. The isolated nucleicacid of claim 13 , wherein the isolated nucleic acid comprises at least200 contiguous nucleotides of SEQ ID NO:5 or its complement.
 17. Theisolated nucleic acid of claim 13 , wherein the isolated nucleic acidcomprises SEQ ID NO:5 or its complement.
 18. An isolated DNA comprisinga strand that comprises a nucleotide sequence that (i) is at least 100nucleotides in length and (ii) hybridizes under highly stringentconditions with SEQ ID NO:4 or 5, or the complement of SEQ ID NO:4 or 5.19. The isolated DNA of claim 18 , wherein the nucleotide sequence is atleast 200 nucleotides in length.
 20. The isolated DNA of claim 18 ,wherein the nucleotide sequence is at least 400 nucleotides in length.21. The isolated DNA of claim 18 , wherein the nucleotide sequence is atleast 1,000 nucleotides in length.
 22. An isolated DNA comprising astrand that comprises a nucleotide sequence that (i) is at least 100nucleotides in length and (ii) shares at least 80% sequence identitywith a fragment of SEQ ID NO:4 or 5 having the same length as thenucleotide sequence.
 23. The isolated DNA of claim 22 , wherein thenucleotide sequence is at least 200 nucleotides in length.
 24. Theisolated DNA of claim 22 , wherein the nucleotide sequence is at least400 nucleotides in length.
 25. The isolated DNA of claim 22 , whereinthe nucleotide sequence is at least 1,000 nucleotides in length.
 26. Ahomologously recombinant cell stably transfected with the DNA constructof claim 1 , the DNA construct having undergone homologous recombinationwith genomic DNA upstream of the ATG initiation codon of an endogenousFSHβ coding sequence.
 27. A homologously recombinant cell stablytransfected with the DNA construct of claim 2 , the DNA construct havingundergone homologous recombination with genomic DNA upstream of the ATGinitiation codon of an endogenous FSHβ coding sequence.
 28. Ahomologously recombinant cell stably transfected with the DNA constructof claim 3 , the DNA construct having undergone homologous recombinationwith genomic DNA upstream of the ATG initiation codon of an endogenousFSHβ coding sequence.
 29. A homologously recombinant cell stablytransfected with the DNA construct of claim 4 , the DNA construct havingundergone homologous recombination with genomic DNA upstream of the ATGinitiation codon of an endogenous FSHβ coding sequence.
 30. A method ofaltering expression of an endogenous FSHβ gene in a mammalian cell, themethod comprising introducing the DNA construct of claim 1 into thecell; maintaining the cell under conditions which permit homologousrecombination to occur between the construct and a genomic target sitehomologous to the targeting sequence, to produce a homologouslyrecombinant cell; and maintaining the homologously recombinant cellunder conditions which permit expression of the FSHβ coding sequenceunder the control of the transcriptional regulatory sequence.
 31. Amethod of delivering FSHβ to an animal, comprising providing the cell ofclaim 26 , and implanting the cell in the animal, wherein the cellsecretes FSHβ.
 32. A method of delivering FSHβ to an animal, comprisingproviding the cell of claim 27 , and implanting the cell in the animal,wherein the cell secretes FSHβ.
 33. A method of delivering FSHβ to ananimal, comprising providing the cell of claim 28 , and implanting thecell in the animal, wherein the cell secretes FSHβ.
 34. A method ofdelivering FSHβ to an animal, comprising providing the cell of claim 29, and implanting the cell in the animal, wherein the cell secretes FSHβ.35. A method of producing FSHβ comprising providing the cell of claim 26, and culturing the cell in vitro under conditions which permit the cellto express and secrete FSHβ.
 36. A method of producing FSH=, comprisingproviding the cell of claim 27 , and culturing the cell in vitro underconditions which permit the cell to express and secrete FSHβ.
 37. Amethod of producing FSHβ, comprising providing the cell of claim 28 ,and culturing the cell in vitro under conditions which permit the cellto express and secrete FSHβ.
 38. A method of producing FSHβ, comprisingproviding the cell of claim 29 , and culturing the cell in vitro underconditions which permit the cell to express and secrete FSHβ.